APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1994, p. 599-605 Vol. 60, No. 2 0099-2240/94/$04.00 + 0 Copyright © 1994, American Society for Microbiology Manganese Peroxidases of the White Rot Fungus Phanerochaete sordida CARMEN RÜTTIMANN-JOHNSON,* DANIEL CULLEN, AND RICHARD T. LAMAR Institute for Microbial and Biochemical Technology, Forest Products Laboratory, Forest Service, U.S. Department ofAgriculture, One Gifford Pinchot Drive, Madison, Wisconsin 53705-2398 Received 7 October 1993/Accepted 4 December 1993 The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene. The lignin-degrading fungus Phanerochaete sordida was first rinated compounds. Evidence that this process also occurs in described by Karsten in 1882 as Corticium sordidum (6) and soil was obtained by Qiu and McFarland (42), who reported was given its present name by Eriksson and Ryvarden (10). that P. chrysosporium enhanced the formation of soil-bound This fungus is found worldwide, especially in the northern residues of the creosote component benzo[a]pyrene. hemisphere, as a saprophyte on hardwood and occasionally on Because (i) fungal extracellular lignin-degrading enzymes softwood, on which it causes white rot decay (6). Previous work may play important roles in pollutant transformation in soil demonstrated that P. sordida isolate HHB-8922-sp (USDA and (ii) there is interest in using P. sordida for soil remediation, Forest Products Laboratory designation) possesses a rapid the present research was undertaken to identify and charac- growth rate over a wide range of temperatures and is superior terize the extracellular lignin-degrading enzymes produced by in the ability to mineralize the wood preservative pentachlo- this fungus. rophenol (PCP) in chemically defined aqueous culture com- pared with P. chrysosporium BKM-F-1767 and several other P. sordida isolates (30). On the basis of these results, this isolate MATERIALS AND METHODS was evaluated further for use in remediation of PCP-contam- Organisms and culture conditions. P. sordida isolate HHB- inated soils. The ability of this fungus to cause rapid and 8922-sp was obtained from the culture collection at the Center extensive decreases in PCP concentrations in both laboratory for Forest Mycology Research at the Forest Products Labora- soils (30) and field soils with (9, 29) and without (28) cocon- tory, Forest Service, U.S. Department of Agriculture, Madi- tamination with creosote demonstrates its potential for use in son, Wis. The fungus was maintained in potato dextrose agar remediation of PCP-contaminated soils. slants at 4°C. All cultures were prepared in the minimal salts Extracellular lignin peroxidases (LiPs), manganese peroxi- medium described by Tien and Kirk (48) but with 10 mM dases (MnPs), and laccases produced by lignin-degrading fungi transaconitic acid as a buffer instead of 2,2-dimethylsuccinate have been shown to catalyze the oxidative degradation of and without veratryl alcohol. All of the experiments described several important pollutants (27). For example, the oxidative were done in shallow cultures consisting of 10 ml of medium in 4-dechlorination of PCP to chloranil (tetrachloro-p- benzoqui- 125-ml Erlenmeyer flasks. They were incubated in stationary none) is catalyzed by both LiP and MnP from P. chrysosporium conditions at 30°C. Beginning on day 3, the flasks were flushed (17). These enzymes have also been shown to be involved in daily with oxygen, unless otherwise stated. All experiments the initial and subsequent oxidations of 2,4-dichlorophenol and were done in triplicate. Blended mycelium was used as the several of its transformation products that lead to complete inoculum. For inoculum preparation, 7-day-old fungal mats mineralization of this compound by P. chrysosporium in aque- were broken up in a Waring blender (five 15-s spurts at high ous culture (49). Fungal laccases, peroxidases, and tyrosinases speed with 30-s intervals to prevent warming of the mycelium). have also been shown to catalyze the in vitro coupling of The cultures were inoculated with 0.45 mg of mycelium chlorophenols to humic materials, yielding chlorophenol-hu- (dry-weight basis) ml-1 of medium. mic material hybrid polymers (44-46), a process that would Optimization of the culture medium for MnP production eliminate the bioavailability and thus the toxicity of the chlo- and search for LiP and laccase. In a series of experiments, different components of the culture medium were varied to * Corresponding author. Mailing address: Institute for Microbial find the best conditions for production of the three extracel- and Biochemical Technology, Forest Products Laboratory, One Gif- lular enzymes. Aliquots of culture supernatant fluids were ford Pinchot Drive, Madison, WI 53705-2398. Phone: (608) 231-9466. taken at different times to assay for activities of MnP, LiP, and Fax: (608) 231-9262. laccase. 599 600 RÜTTIMANN-JOHNSON ET AL. APPL. ENVIRON. MICROBIOL. To study the effect of the nitrogen concentration, we pre- protein liquid chromatography system. Potassium phosphate pared cultures containing 1.0 and 10.0 mM ammonium tartrate buffer (50 mM, pH 7.0) containing 1.7 M ammonium sulfate and a non-growth-limiting concentration of the carbon source was used as the mobile phase at a flow rate of 0.5 ml min-1. (1% glucose). These cultures contained 11 µg of Mn(II) ml-1. The proteins were eluted with a gradient of ammonium sulfate Mn(II) was always added as its sulfate form (MnSO4). All of that varied from 1.7 to 0.85 M in the first 20 min and from 0.85 the concentrations given are for the free ion and not the salt. to 0.0 M in the following 40 min. The MnP peaks obtained The effect of oxygen was studied by flushing cultures con- from this chromatographic step were pooled, concentrated, taining 1.0 mM ammonium tartrate and 11 µg of Mn(II) ml-1 and dialyzed. daily with either 100% oxygen or air, beginning on day 3 after Characterization of MnP isozymes. The molecular weights inoculation. of the three isozymes were determined by sodium dodecyl The cultures used to assess the effect of Mn(II) contained sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) per- 0.3, 11.0, 40.0, or 100.0 µg of Mn(II) ml-1 in standard medium formed with the Phast System (Pharmacia). Gels (10 to 15%) with 1.0 mM ammonium tartrate and an oxygen atmosphere. and conditions recommended by the manufacturer were used. The effect of pH was studied by using 10 mM transaconitic acid as a buffer at pHs 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0. The Staining was done with Coomassie blue. The molecular size cultures contained 1.0 mM ammonium tartrate and 11 µg of standards used were rabbit muscle phosphorylase b (97.4 kDa), Mn(II) ml-1. bovine serum albumin (66.2 kDa), chicken egg white ovalbu- The effect of veratryl alcohol on LiP production was assayed min (45 kDa), bovine carbonic anhydrase (31 kDa), soybean by adding a 0.4 mM concentration of the alcohol to cultures trypsin inhibitor (21.5 kDa), and chicken egg white lysozyme that contained 1.0 mM ammonium tartrate and 11 µg of (14.4 kDa). -1 MnSO4 ml . The isoelectric points of the proteins were determined with Cultures designed to enhance laccase production contained a Bromma 2117 Multiphor apparatus (LKB) and Servalyt 10 mM MES (morpholineethanesulfonic acid) buffer (pH 6.5) Precoats polyacrylamide gels (Serva Fine Biochemicals Inc., in the presence of 500 µg of CuSO4 per liter (15). Also, the Westbury, N.Y.) with a pH range of 3 to 6. Proteins were laccase inducers p-hydroxybenzoic acid (1 mM) (15) and focused for 5 min at 400 V and then for 5 min at 800 V. The 2,5-xylidine (0.2 mM) (1 1) were added to cultures made in the standards used were amyloglucosidase (pI 3.55), trypsin inhib- standard conditions described previously. itor (pI 4.55), ß-lactoglobulin A (pI 5.13), and bovine carbonic The production of enzymes in shaking cultures was also anhydrase (pI 5.85). Proteins were stained with Serva Violet. assessed. Cultures were made in 125-ml flasks containing 30 ml MnP activity staining was done with 4-chloro-1-naphthol as the of culture medium and shaken at 180 rpm. Tween 20 (0.1 or substrate. Five milligrams of the substrate was dissolved in 1.7 0.5%) was also added to these cultures. ml of methanol and mixed with 8.3 ml of 20 mM Tris-HCl (pH Enzyme assays. MnP activity was assayed by using vanilly- 7.5)-0.5 M NaCl. Eight milliliters of the resulting solution was lacetone as the substrate, as described by Paszczynski et al.